J Appl Phycol DOI 10.1007/s10811-017-1249-5
Chemodiversity of the brown algae Canistrocarpus cervicornis (Dictyotaceae, Phaeophyceae) in tropical and subtropical populations along the southwestern Atlantic coast of Brazil Juliana Magalhães de Araujo 1 & Marcelo Raul Romero Tappin 2 & Rafael da Rocha Fortes 3 & Erick Alves Pereira Lopes-Filho 1 & Fabiano Salgueiro 1 & Joel Campos De Paula 1
Received: 23 February 2017 / Revised and accepted: 15 August 2017 # Springer Science+Business Media B.V. 2017
Abstract Three populations of the brown seaweed Canistrocarpus cervicornis along 1800 km of the southwestern Atlantic coast were studied. Phylogenetic analysis based on the plastid and mitochondrial genes rbcL and nad1, respectively, confirmed that these populations were composed of the same taxon. Crude ethyl acetate extracts were analysed by gas chromatography coupled to a mass spectrometer (GC/MS), which revealed 13 detectable products whose fragment patterns were compatible with those expected for diterpenes. The analysis of fragment patterns identified the diterpenoid 4,7diacetoxy-14-hydroxydolastane-1 (15), 8-diene as the major product in all the populations. Furthermore, the concentration of this diterpenoid and other products were highly variable among the three populations. However, this plasticity was not observed in sample morphology, which was generally conserved among the populations. Therefore, GC/MS enabled the detection of differences in diterpene chemodiversity within geographically distinct populations of C. cervicornis, and even though natural product abundance varied greatly, molecular markers and morphology were highly conserved.
Electronic supplementary material The online version of this article (doi:10.1007/s10811-017-1249-5) contains supplementary material, which is available to authorized users. * Joel Campos De Paula
[email protected] 1
Programa de pós-graduação em Biodiversidade Neotropical, Universidade Federal do Estado do Rio de Janeiro, Av. Pasteur 458, 409, Rio de Janeiro 22290-255, Brazil
2
Fundação Oswaldo Cruz, Rede de Plataformas Tecnológicas, Av. Brasil, 4365 - Manguinhos, Rio de Janeiro 21040-900, Brazil
3
Instituto de Biologia, Universidade Federal do Estado do Rio de Janeiro, Av. Pasteur 458, Rio de Janeiro 22290-255, Brazil
Keywords Chemodiversity . rbcL . nad1 . GC/MS . Diterpene . Phylogeny
Introduction Chemical studies have demonstrated that natural products from the algal family Dictyotaceae are rich in metabolites and represent an important source of biologically active diterpenes (De Paula et al. 2011). Such diterpenes have been associated with a variety of functions in the marine environment, including defence and chemical signalling (Fleury et al. 1994; Bianco et al. 2009; Bianco et al. 2010; Plouguerné et al. 2010; Paul et al. 2011; Marty and Pawlik 2015), and possess cytotoxic, antiviral and antibiotic bioactivities that could be used for scientific research (Cavalcanti et al. 2011; De Paula et al. 2011). Much like molecular sequences, which have revolutionised macroalgae systematics, starting in the 1990s (Saunders and Druehl 1992), natural products can also be used as taxonomic markers (Kelecom and Laneuville Teixeira 1986; De-Paula et al. 2008). The description of the genus Canistrocarpus De Paula et De Clerck, for example, is based on three distinct types of data: molecular (species are genetically distant from other related genera), morphological (species possess a welldeveloped involucrum of sterile cells that surround the sporangia and a pigmented multicellular paraphyses that surrounds the antheridia) and chemical (species possess group IIb diterpenes, including dolastanes and seco-dolastanes). The genus comprises Canistrocarpus crispatus (Lamouroux) De Paula et De Clerck, C. magneanus (De Clerck et Coppejans) De Paula et De Clerck and C. cervicornis (Kützing) De Paula et De Clerck (De Clerck et al. 2006), the last of which possesses a transoceanic (~cosmopolitan) distribution and is
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found all along the Brazilian coast (Széchy and De Paula 2016). Studies on such cosmopolitan species can offer insights into the evolutionary and ecological processes of the marine benthic environment (Masuda et al. 1997; Boo et al. 2014), such as distinguishing between recent and historical events; low genetic distance between distant populations is generally related to contemporary dispersal or invasive processes, whereas high genetic distance reflects historical events that have caused discontinuities among populations and geographical isolation (Fraser et al. 2010). For this purpose, the present study collected C. cervicornis samples from three locations along 1800 km of the Brazilian coast and analysed using gas chromatography coupled to a mass spectrometer (GC/MS), in order to obtain the natural products commonly used for chemotaxonomic purposes (Amico 1995; De-Paula et al. 2007; Le Lann et al. 2008; Le Lann et al. 2014; Farias de Mesquita et al. 2015). Additionally, the specimens were identified and the genetic diversity was evaluated by sequencing the plastid and mitochondrial genes rbcL and nad1, respectively. The present study also measured variation in the morphological, chemical and molecular characteristics of the C. cervicornis specimens in relation to their biogeographical locations. The study area covered ~ 14 degrees of latitude, 1800 km of the southwestern Atlantic coast, and both tropical and warmtemperate regions (Spalding et al. 2007). The southernmost population can be considered an ecotone between the tropical region to the north and the warm-temperate region to the south. The transition area between these ecoregions is recognised as a biogeographic barrier for most of the macroalgae flora from the western South Atlantic coast (Brasileiro et al. 2009) but also possesses a high diversity of organisms with tropical and temperate affinities (Guimaraes and Coutinho 1996).
Materials and methods Sampling The C. cervicornis specimens were collected during the winter (end of June to the end of September) of 2012 from three locations along the coast of Brazil (western South Atlantic): (1) Prainha Beach (22° 57′ 6′′ S, 42° 0′ 14′′ W), city of Arraial do Cabo, Rio de Janeiro state (RJ); (2) Penha Beach (12° 59′ 27′′ S, 38° 37′ 29′′ W), city of Vera Cruz, Bahia state (BA) and (3) Muro Alto Beach (08° 25′ 45′′ S, 34° 58′ 40′′ W), city of Ipojuca, Pernambuco state (PE; Table S2). The locations were separated by ~1380 km (locations 1 and 2) and 475 km (locations 2 and 3) and spanned a total distance of ~1800 km. The algae were collected from depths of 1–2 m using SCUBA. Each individual was separated into three sub-samples. The first and second sub-samples were preserved in silica gel for subsequent chemical and molecular analysis, respectively,
whereas the third sub-sample was preserved in 4% formalin for taxonomic studies and was later deposited in the herbarium of the Universidade Federal do Estado do Rio de Janeiro (HUNI). Data about herbivores on sampling areas was surveyed on online databases (e.g. FishBase). Morphological analysis Morphological analysis was performed on 15 individuals from each population, according to Tronholm et al. (2010). The analysis included measuring the thallus (length and width); taking cell measurements from the cortex and medulla of histological sections of the apical, middle and basal parts of the thallus, using an Olympus CX40 light microscope (Tokyo, Japan) under ×100 magnification and recording colour, type of branching, apex form, presence of proliferations and life stage, using an Olympus SZ51 stereoscopic microscope (Tokyo, Japan). Chemical analysis Extraction Twenty-milligramme samples of dry mass from each individual (three individuals per population) were separately macerated in 2-mL microtubes, and 1.5 mL ethyl acetate (C4H8O2) was added to each sample, after which the mixtures were placed in an immersion ultrasound for 10 min at room temperate (25 °C). The resulting extracts were filtered through a regenerated cellulose membrane (Minisart RC15, Sigma-Aldrich, Brazil) with 0.45-μm mesh. The colour of the crude extracts varied from light green to olive green. GC/MS analysis The filtered extracts were analysed using a gas chromatograph (Agilent model 6890n) that was coupled to a mass spectrometer (model 5973n) with an automated injector (model 7683) and a db-5 ms column (30 m, 0.250 mm internal diameter, 0.250-μm film thickness). Hydrogen was used as the carrier gas, with a flow rate of 1.3 mL/min-1, and the samples were injected with a split ratio of 1:10. The initial temperature was 141 °C, and the samples were heated at a rate (slope) of 2.74 °C min−1, until reaching 278 °C, where the samples were held for 50 min. Meanwhile, the temperatures of the injector and detector were fixed at 270 and 325 °C, respectively. Four replicates were performed for each extract, and three extracts were analysed per population (i.e. RJ, BA and PE). The resulting chromatograms and its fragmentation patterns were analysed using Open Chrome, in order to detect the products of interest from each individual and population, and the resulting mass spectra were compared with those in the Wiley Library (~229,000 spectra). Data processing The areas of peaks in the crude extract chromatograms were interpreted as the relative abundance of the corresponding diterpenes, and the mean (n = 3) peak area
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corresponding to each product was subject to principal component analysis (PCA) using Primer 6 (version 6.1.13), in order to highlight differences between the populations. In addition, significant differences in the product abundance of the populations were verified using analysis of variance (Dunn’s test) in SigmaPlot (v. 11; Supplementary Table S3). Only the 13 most abundant products from each population were used in this analysis because the other products could not be clearly distinguished from the baseline of the chromatograms. Molecular analysis Total DNA was extracted from the same individuals used for chemical analysis following a modified version of the 2X CTAB method, as described by De Clerck et al. (2001). Two genes, the mitochondrial NADH dehydrogenase subunit 1 (nad1) and the plastidial ribulose-bisphosphate-carboxylase/ oxygenase (rbcL), were amplified using polymerase chain reaction (PCR), according to Tronholm et al. (2010), and sequenced by Macrogen Inc., Korea. The editing, alignment and phylogenetic analysis of the resulting sequences were performed using MEGA6 (Tamura et al. 2013). The maximum likelihood (ML) analysis included 13 sequences (Table S4) and used the Kimura 2-parameter nucleotide substitution model and nearest-neighbour interchange (NNI) heuristic search method, with 1000 bootstrap replicates.
Results The GC/MS spectra of the crude extracts revealed 13 main products that were shared among all the individuals sampled, thereby confirming the importance of natural products as taxonomic markers (Kelecom and Laneuville Teixeira 1986). The same major product, 4,7-diacetoxy-14hydroxydolastane-1 (15), 8-diene (product 9), which had a retention time of 33.36 min (Table 1), was found in all three populations (Fig. 1) and was confirmed by comparing the fragmentation patterns with data in the literature (Oliveira et al. 2008; Bianco et al. 2010; Sun et al. 1981; González et al. 1983). Product 7, which had a retention time of 29.32 min, was the second most abundant product in all the populations, and the average relative abundance of the two major products in the PE population was significantly different (p < 0.01) than that of the same products in the other two populations. The relative abundances of products 9 and 7 in the PE population were ~ 2.8 and ~ 8.1 times greater than those in the BA population, which was located 475 km southward, and 2.5 and 2.9 times greater than those in the RJ population, which was located 1800 km southward (Fig. 2). Meanwhile, the other products (1–6, 8 and 10–13) were only found in small quantities or trace amounts (Table 1). The mass spectra and fragmentation
Table 1 Canistrocarpus cervicornis diterpenes ranked by its retention time (RT) and its relative abundance (the sum of the abundance of the representative products = 100%)
Product
RT
RJ
Ba
Pe
1 2
23.06 27.52
t o
t t
t o
3 4
27.80 27.97
t t
t t
t t
5
28.20
o
t
t
6
28.77
o
o
t
7 8
29.32 32.88
p o
p t
p t
9 10
33.36 33.56
m t
m t
m t
11
34.16
a
t
p
12 13
34.44 35.11
o t
t t
t o
The most abundant products are italicized RT retention time of the products, in minutes; m majority (> 40%); a abundant (20–40%); p present (20–8%); o occasional (2–7%); t trace (< 2%)
patterns of the most abundant diterpenes are presented in Fig. S1 and Table S1, respectively, and the overlap of chromatograms in Fig. S2. The PCA indicated that the products of the populations were not equal, mainly in regards to the major products (7 and 9). The relatively high abundance of these two products influenced the position of the tropical populations along axis 1 of the PCA (Fig. 3). Although the distance between the PE and BA populations is less than 500 km, the PE population possessed the highest relative abundances of products 9 and 7, whereas the BA population possessed the lowest relative abundances. In addition, axis 1 was positively influenced by the abundance of these two products, whereas axis 2 was positively influenced by the relative abundance of product 11 (retention time 34.158 min), which was abundant in the RJ population (Table 1 and Supplementary data). The RJ population was unique in that the relative abundance of a large
Fig. 1 Chemical structure of diterpene 4,7-diacetoxy-14hydroxydolastan-1 (15), 8-diene, proposed by Bianco et al. 2010
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Fig. 2 Map of Canistrocarpus cervicornis populations along the Atlantic coast of Brazil. Corresponding chromatograms are superimposed, along with the diversity and abundance of natural products identified (retention
times between 27.4 and 35.15 min). PE Pernambuco (northern tropical population), BA Bahia (southern tropical population), RJ Rio de Janeiro (warm-temperate ecotone)
number of products was distinct from that of the other two populations (Table 1 and Fig. 3). The morphological analysis revealed that the specimens from all three populations were morphologically similar, and
no gametophytic thalli were observed, even though they had been previously reported by Ateweberhan et al. 2005, who suggested that the absence of gametophytic thalli could imply that populations depend on asexual reproduction, probably by
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Discussion
Fig. 3 Principal component analysis (PCA): Discriminatory projection of the products encountered in the three populations sampled. PE Pernambuco (northern tropical population), BA Bahia (southern tropical population), RJ Rio de Janeiro (warm-temperate ecotone)
the fragmentation of vegetative thalli, in situ germination of spores and detachment of surface proliferations, which would result in consecutive sporophyte populations without the occurrence of gametophytes. The nad1 (mitochondrial) and rbcL (chloroplastidial) genes and their p distances were used to confirm the identity of specimens as it is indicated on the phylogenetic tree for rbcL (Fig. 4). There were no significant differences between the 686-bp nad1 matrices of the C. cervicornis populations from the southwestern Atlantic (Brazil) and northeastern Atlantic (Canary Islands; p distance = < 0.001), nor were there any differences between the 1553-bp rbcL matrices of the populations from the southwestern Atlantic (Brazil) and Philippines (p distance = 0.002) or the populations from Brazil and Tanzania (p distance = 0.005). These genes have been used to distinguish between other members of the Dictyotales (De Clerck et al. 2006; Bittner et al. 2008; Tronholm et al. 2010; Silberfeld et al. 2014). For example, C. cervicornis and C. crispatus are genetically separated, as indicated by the p distances between their rbcL and nad1 sequences (0.008 and 0.085, respectively), and Dictyota dichotoma, the type species of the genus Dictyota, and C. cervicornis are separated by p distances of 0.1 and 0.2, based on rbcL and nad1 sequences, respectively. For the Brazilian populations, there were no significant differences between the populations, based on either of the genes, except for a single nucleotide substitution in the nad1 sequence of a single specimen from the PE population; however, the region where this sample was located was not included in the analysis. Therefore, the genes studied in C. cervicornis are conserved in relation to environmental variation and geographical distance.
Many studies have attempted to elucidate the chemical variation among Canistrocarpus and Dictyota populations but have lacked robust information because their evaluations were based on extracts obtained from aggregates of many individuals (e.g. Oliveira et al. 2008; De Paula et al. 2008; OrtizRamirez et al. 2008; Freitas et al. 2007). The present study refined this type of analysis by measuring the relative abundance of products in extracts of individual specimens and detected 13 most abundant diterpenes from C. cervicornis using their mass spectra and available literature (Oliveira et al. 2008). Both the molecular and morphological analyses confirmed that the three populations are C. cervicornis. The RJ population belongs to an ecotone because it borders the tropical and warm-temperate zones and, therefore, is influenced by greater environmental variation and probably the greater relative abundance of other products (Kark and van Rensburg 2007). The data from the present study support this hypothesis, although a clear pattern of increasing relative abundance of the major product from the south to the north was not observed. The synthesis of natural products can be quantitatively dynamic and can be influenced by environmental factors, whereas the qualitative variability of products (i.e. types of products present) tends to be more conservative (Masuda et al. 1997). In the present study, the sample locations varied in their wave dynamics, salinity, depth of the specimen collection, and turbidity (Table S2), all of which could have contributed to the observed chemical plasticity. The temperature difference could also support the hypothesis that environmental conditions contribute to variation in the abundance of natural products between locations as described under culture conditions by Sudatti et al. (2011) for Laurencia. The Arraial do Cabo region (RJ) is influenced by an upwelling phenomenon, where coastal waters are driven seaward by winds and then replaced by upwelling of South Atlantic Central Water (SACW), which significantly reduces the water temperature during the spring and summer (Castelão et al. 2004). In the present study, samples were collected from all of the locations during the winter, in order to minimise these effects. During winter in Arraial do Cabo, the intensity of the northeastern winds diminishes, and the SACW retreats towards the shelf margin, which homogenises the coastal zone, with water temperatures ranging from 20 to 25 °C. As the three populations had the same product richness, the higher diversity should be attributed to that had the more equitable abundance among the products. So, the RJ population yielded a high diversity of natural products, with 41% represented by the major product and the remaining 59% distributed across the other 12 products. In the other populations, the major product made up more than half of the total products (84% in the BA population and 57% in the PE population), a difference that could be the result of the higher
J Appl Phycol Fig. 4 Phylogenetic tree of rbcL gene sequences. Topology obtained by maximum likelihood. Values at nodes indicate bootstrap values (1000 replicates)
temperatures found at these locations. According to Joshi and Gowda (1975), temperature can influence the secondary metabolism of macroalgae, and, for this reason, the influence of the SACW could be perpetuated in local populations, even during intermediate periods between individual phenomena, and, in relation to the secondary metabolism of C. cervicornis, could be reflected in the higher diversity of products and, consequently, the higher abundance of products (Fig. 2). Natural products perform a great variety of functions, including defence (Hay 2012), that can be associated with herbivory pressure. To test this hypothesis, the present study analysed the correlation between the total abundance of the natural products and herbivorous fish that occur on the continental shelf (bathymetry to 200 m deep). The family Pomacentridae is often associated to rocky shores and reefs and is the richest group of fishes at depths where C. cervicornis grows. The PE population, which yielded the highest relative abundance of total products and the major product, was associated with greater fish species richness than the other two population regions. Even though the BA population possessed a similarly rich assemblage of herbivorous fishes (mainly Pomacentridae), the abundance of products was approximately three times less than that of the PE population. The high relative abundance of the major product in the BA population is remarkable, in that it represents 84% of the total products, which may indicate that the substance is particularly effective in deterring herbivorous fish in the tropical zone. Rhoades (1979) postulated that organisms produce defences in direct proportion to the risk of predation, in a relation of cost-benefit (Optimal Defence Model). In the presence of predators, the level of defence can increase and be targeted, which could explain the variation among populations and among individuals from the same populations (Cronin 2001). Hay and Steinberg (1992) presented the ‘Theory of Spatial Variation of Consumers’, which predicts that plants with chemical defences are evolutionarily more persistent in areas with higher herbivory than in areas where herbivory is less prevalent. Moreover, macroalgae from environments with
high herbivory pressure are potentially attacked more often and, for this reason, are better equipped for defence. Similarly, among the three populations, the RJ population possessed a lower abundance of the major product (two times less) than the PE population and a greater variety of products in higher concentrations. This population may not be subject to predation by a diverse fish assemblage but is subjected to wider variation in environmental conditions (i.e. temperature) and herbivory by invertebrates. Another factor that would generate differences among populations, from a chemical point-of-view, would be the specimen’s maturity or life stage. However, in the present study, only adult sporophytes were sampled. The GC/MS analysis of the present study revealed the qualitative and quantitative chemodiversity of diterpenes in geographically distinct populations of C. cervicornis. Although the abundance of the natural products varied greatly among the three populations, there was no variation in morphology of the populations or the corresponding mtDNA and cpDNA gene sequences. Therefore, differences in the chemical profiles of the populations are likely due to differences in environmental conditions. Acknowledgments The authors thank Roberta Pacheco Silva and Samara Ribeiro da Silva for their help in the molecular biology laboratory, Dr. Silvia Mattos Nascimento for the comments that improved our manuscript, and the Program for Technological Development in Tools for Health-PDTISFIOCRUZ (Fundação Oswaldo Cruz) for use of its facilities. This work was supported by the Fundação Carlos Chagas Filho de Apoio à Pesquisa do Estado do Rio de Janeiro (BIOTA 2012 – FAPERJ E-26/111.397/2012) and a Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) scholarship to JMA.
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